Project Summary/Abstract To initiate molecular characterization of the calcium-activated chloride channels (CaCCs) that have been found in multiple neuronal types since 1980s, we first showed that CaCC is formed by TMEM16A or TMEM16B of the mammalian TMEM16 family of ten members in 2008. To ask how CaCC works, we investigated the calcium gating mechanism. First, we showed that a fruit fly homolog of the TMEM16 family, which we named Subdued, forms CaCC. Next, we mutated all 38 acidic residues that are evolutionarily conserved in fruit fly and mammalian CaCCs, to identify five acidic residues that strongly impact the calcium sensitivity of CaCC. After reporting our study, we are pleased to see that these five acidic residues correspond to the five acidic residues that bind two calcium ions in the recently reported structure of the fungal TMEM16 homolog, nhTMEM16. We will continue with our biophysical studies to elucidate how CaCC works by combining structural analyses of TMEM16A via single-particle electron cryo-microscopy (cryo-EM) with site- directed mutagenesis and electrophysiological studies. Having found CaCC involvement in the modulation of the action potential waveform and excitatory synaptic potentials in hippocampal neurons as well as action potential firing of inferior olivary neurons and cerebellar motor learning, we aim to conduct mechanistic studies to determine how CaCC works, in order to better understand CaCC modulation of neuronal signaling: Whereas it is well known that CaCC channel activity leads to membrane potential change, it is an intriguing open question as to how voltage across the membrane affects CaCC function. Whereas we know CaCC is activated by elevation of intracellular calcium that may result from calcium influx through calcium channels or NMDA receptors or calcium release from internal stores, it is unknown whether CaCC activation at low or high internal calcium concentration, which likely reflects different physiological contexts for CaCC modulation in neurons, might lead to the opening of different permeation pathways for chloride ions. Moreover, it is important to understand how chloride and other permeant ions such as iodide might exert feedback regulation of CaCC activity. Mechanistic understanding of CaCC function and modulation at the molecular level will not only provide insight as to how CaCC fulfills its physiological functions in the brain but also facilitate future development of CaCC modulators of potential therapeutic values.